한국산 다람쥐(tamias sibiricus asiaticus, gmelin)의 실험적 결핵증에 관한 연구

Abstract

[한글]

Experimental Tuberculosis on Korean Chipmunk(Tamias sibiricus asiaticus, Gmelin)

Hoon-Young Chang, M.D.

Department of Medical Science The Graduate School, Yonsei University

(Directed by Prof. Joon Lew, M. D., Ph.D.)

A dramatic successes have followed one another in the discovery of substances

curative in most acute and some chronic bacterial infections, attention bas been

focused on those infections where such success has not been so complete. One such

infection is tuberculosis. Although the discovery of streptomycin, isoniazid and

para-aminosalcylic acid have provided the drugs of the greatest importance, moot

investigators and clinicians feel that these primary antituberculosis drugs are not

the final word in the therapy of this infection.

Despite its widespread use in the study of chemotherapy of many exerimental

diseases, the mouse, guinea pig and rabbit have been extensively used for

therapeutic evaluations in experimental model of tuberculosis in recent years. Much

of this work has recently been reviewed by Raleigh and Youmans and detailed

reference to it need not be made here, except perhaps to put additional emphasis on

differences in various strains in their susceptibility to experimental infection

with this agent (tuberculosis bacilli).

The success of any screening program depends to a considerable extent upon the

choice of an appropriate test animal. Ideally, the animal should be relatively

inexpensive, small enough to be conveniently cared for and sufficiently sensitive

to the particular infectious agent as to die from the infection or at least to

present easily detectable diagnostic criteria. The disease should develop within a

reasonably short incubation period after inoculation and the gross pathological

changes should be sufficiently distinct and characteistic to lend them selves to a

numerical appreciation of the extent of infection and of protection afforded by a

drug.

The animal infection used in drug screening should respond to chemotherapy in the

same manner as could be expected in the human disease iota which a drug is sought.

The significans screening results is much improved when not only one, but a number

of different laboratory teste are used simultaneously.

In these respects the present author has been led to abandon the above known

laboratory animals in favor of the chipmunk for the fellowing reasons. Work with

chipmunks in tuberculosis and other infections has shown the importance of the use

of highly susceptible animals when few animals can he tested and uniformity of

response is desirable. No ready source of highly susceptible and uniformity of

responsive guinea pigs or mile art available while on the other hand, such source

of single strain of chipmunk are readily available. Finally, there is no evidence

that the results of chemotherapy in tuberculosis of the guinea pigs or mice are

more certain guide to the usefulness of a given compound in man than are the

results obtained in chipmunks.

A consideration of these facts has led us to an investigation of the use of the

chipmunks for invitro tests for experimental tuberculosis.

The present paper is offered as a contribution to the knowledge of chipmunks

tuberculosis as furnished by delineation of the bacterial distributions and of the

higtopathology producedunder the conditions of these experiments. Materials and

methods are as fellows.

Cultures: Two strains of Mycobacterium tuberculosis var, hominis H^^37 RV and

var, bovis, Ravened, were used in the present study. Both strains were obtained

from the National Institute of Health in Korea and were maintained in the

Department of Microbiology, Yonsei University, College of Medicine, Seoul.

Medium: Stock cultures were kept and stored at 3 to 5℃ on the Ogawa's and

Loewengtein-Jenaen's media.

Cultures to be used for chipmunk arid mouse injection were grown freshly on the

above solid medium about for 3 weeks. A few flakes of vigorously grown tubercle

bacilli are placed on the sterile filter paper and weighed actual wet weight of

baotria and placed carefully in the bottom of a sterile agate motor. These are

ground by hand for 15 to 20 minutes until a relative]y homogenous suspension is

obtained.

This is diluted to a weight of 2 mg per ml by the gradual addition of sterile

distilled water. The resulting suspension is then transferred to a sterile vial and

kept in the refrigerator until inoculatiom being started.

The suspensions are diluted so as to contain the desired inoculum for each tube

in adequate amount of phosphate buffer or distilled water. Then animals under study

were given an intraperitoneal, subcutaneous and intramuscular inoculation of the

diluted culture.

Aninals: The chipmunks (Tamias sibiricus asiaticus, Gmelin) were obtained from

market and maintained on a nutritional diet and carefully observed their

adaptability to the confined cage for 2 months prior to inoculate the bacterial

suspension. White CFW mice were also obtained from market and were used in all of

the control studies.

Animal inoculation: H^^37 Rv and Ravenel strains were appropriately diluted for

use as procedures inocula as mentioned above. The chipmunks used in transmission

were injected subcutaneously,intraperitoneally and intramuscularly with 2 mg, per

each groups.

The mice, control animals, were injected with 0.5 mg of wet weight of bacilli

through the same routes.

Observations: As soon as possible after death, immediately in the case of those

sacrificed, the animals were eviscerated and notations made of the extent of gross

pathology present in the various organs and weighed each of them.

At this time half of the organs specimen was removed for the bacterial count and

rest viscera were then fined 10% formaldehyde solution. These were re-examined in

detail in order to form a quantitatative and qualitative appreciation of the gross

pathology, and the relative degree of change in these organs. Tissues were

dehydrated and paraffin embeded according to the usual technic, placing slices from

all of the organs of each animal in a single block, and sections cut at 5 microns.

Each was stained with hematoxylin and eosin and by the Ziehl-Neelsen carbol fuchsin

method for acid fast organisms. The extent of microscopic involvement of a given

organ was tabulated on the fellowing basis; Zero (0), one plus (+), mild chronic

granulomatous infiltrations observed more than half of the fields; two plus (++),

moderate chronic granulomatous infiltrations; three pius (+++), severe chronic

granulomatous infiltrations. These methods for the estimation of the accocunt of

tuberculous involvement differ slightly from those used by Youmans and McCarter or

by Raleigh and Youmans.

The spleen, liver, lungs, kidneys and lymph nodes were measured by torsion

balance immediately after eviscerated and especially the spleen index (S.I.) was

calculated by the formula of the root of spleen weight by 100/body weight.

Conclusion

The Korean chipmunks, (Tamias sibiricus asiaticus, Gmelin) were studied on the

susceptibility to the tubercle bacilli and the probability of experimental animal.

The results are as fellows:

1. The chipmunks are more susceptible to the tubercle bacilli than the mice.

2. The chipmunks are more susceptible to the strain H^^37 Rv than the Ravenel.

3. In the chipmunks, extensive infectivity are observed in the spleen, liver and

lymph nodes.

4. In the chipmunks infected with bacilli (H^^37 Rv and Ravenel), the macroscopic

findings, bacterial number, Gaffky index, and histopathological findings in the

spleen, liver and lymph nodes show the parallel relationship.

5. In the chipmunks infected with H^^37 Rv and Ravenel, 50 percent letal days are

22.3 days and 21 days respectively.

6. The chipmunks is most susceptible to tubercle bacilli among the known animal

to date, and able to feed as an experimental animal and appropriate to an

experimental animal for experiulental toberculosis.

[영문]

A dramatic successes have followed one another in the discovery of substances curative in most acute and some chronic bacterial infections, attention bas been focused on those infections where such success has not been so complete. One such infection is tuberculosis. Although the discovery of streptomycin, isoniazid and para-aminosalcylic acid have provided the drugs of the greatest importance, moot investigators and clinicians feel that these primary antituberculosis drugs are not the final word in the therapy of this infection.

Despite its widespread use in the study of chemotherapy of many exerimental diseases, the mouse, guinea pig and rabbit have been extensively used for therapeutic evaluations in experimental model of tuberculosis in recent years. Much of this work has recently been reviewed by Raleigh and Youmans and detailed

reference to it need not be made here, except perhaps to put additional emphasis on differences in various strains in their susceptibility to experimental infection with this agent (tuberculosis bacilli).

The success of any screening program depends to a considerable extent upon the choice of an appropriate test animal. Ideally, the animal should be relatively inexpensive, small enough to be conveniently cared for and sufficiently sensitive to the particular infectious agent as to die from the infection or at least to present easily detectable diagnostic criteria. The disease should develop within a reasonably short incubation period after inoculation and the gross pathological changes should be sufficiently distinct and characteistic to lend them selves to a numerical appreciation of the extent of infection and of protection afforded by a drug.

The animal infection used in drug screening should respond to chemotherapy in the same manner as could be expected in the human disease iota which a drug is sought. The significans screening results is much improved when not only one, but a number of different laboratory teste are used simultaneously.

In these respects the present author has been led to abandon the above known laboratory animals in favor of the chipmunk for the fellowing reasons. Work with chipmunks in tuberculosis and other infections has shown the importance of the use of highly susceptible animals when few animals can he tested and uniformity of response is desirable. No ready source of highly susceptible and uniformity of responsive guinea pigs or mile art available while on the other hand, such source of single strain of chipmunk are readily available. Finally, there is no evidence that the results of chemotherapy in tuberculosis of the guinea pigs or mice are more certain guide to the usefulness of a given compound in man than are the results obtained in chipmunks.

A consideration of these facts has led us to an investigation of the use of the chipmunks for invitro tests for experimental tuberculosis.

The present paper is offered as a contribution to the knowledge of chipmunks tuberculosis as furnished by delineation of the bacterial distributions and of the higtopathology producedunder the conditions of these experiments. Materials and methods are as fellows.

Cultures: Two strains of Mycobacterium tuberculosis var, hominis H^^37 RV and var, bovis, Ravened, were used in the present study. Both strains were obtained from the National Institute of Health in Korea and were maintained in the

Department of Microbiology, Yonsei University, College of Medicine, Seoul.

Medium: Stock cultures were kept and stored at 3 to 5℃ on the Ogawa's and Loewengtein-Jenaen's media.

Cultures to be used for chipmunk arid mouse injection were grown freshly on the above solid medium about for 3 weeks. A few flakes of vigorously grown tubercle bacilli are placed on the sterile filter paper and weighed actual wet weight of baotria and placed carefully in the bottom of a sterile agate motor. These are ground by hand for 15 to 20 minutes until a relative]y homogenous suspension is obtained.

This is diluted to a weight of 2 mg per ml by the gradual addition of sterile distilled water. The resulting suspension is then transferred to a sterile vial and kept in the refrigerator until inoculatiom being started.

The suspensions are diluted so as to contain the desired inoculum for each tube in adequate amount of phosphate buffer or distilled water. Then animals under study were given an intraperitoneal, subcutaneous and intramuscular inoculation of the diluted culture.

Aninals: The chipmunks (Tamias sibiricus asiaticus, Gmelin) were obtained from market and maintained on a nutritional diet and carefully observed their adaptability to the confined cage for 2 months prior to inoculate the bacterial suspension. White CFW mice were also obtained from market and were used in all of

the control studies.

Animal inoculation: H^^37 Rv and Ravenel strains were appropriately diluted for use as procedures inocula as mentioned above. The chipmunks used in transmission were injected subcutaneously,intraperitoneally and intramuscularly with 2 mg, per each groups.

The mice, control animals, were injected with 0.5 mg of wet weight of bacilli through the same routes.

Observations: As soon as possible after death, immediately in the case of those sacrificed, the animals were eviscerated and notations made of the extent of gross pathology present in the various organs and weighed each of them.

At this time half of the organs specimen was removed for the bacterial count and rest viscera were then fined 10% formaldehyde solution. These were re-examined in detail in order to form a quantitatative and qualitative appreciation of the gross

pathology, and the relative degree of change in these organs. Tissues were dehydrated and paraffin embeded according to the usual technic, placing slices from all of the organs of each animal in a single block, and sections cut at 5 microns.

Each was stained with hematoxylin and eosin and by the Ziehl-Neelsen carbol fuchsin method for acid fast organisms. The extent of microscopic involvement of a given organ was tabulated on the fellowing basis; Zero (0), one plus (+), mild chronic

granulomatous infiltrations observed more than half of the fields; two plus (++), moderate chronic granulomatous infiltrations; three pius (+++), severe chronic granulomatous infiltrations. These methods for the estimation of the accocunt of tuberculous involvement differ slightly from those used by Youmans and McCarter or by Raleigh and Youmans.

The spleen, liver, lungs, kidneys and lymph nodes were measured by torsion balance immediately after eviscerated and especially the spleen index (S.I.) was calculated by the formula of the root of spleen weight by 100/body weight.

Conclusion

The Korean chipmunks, (Tamias sibiricus asiaticus, Gmelin) were studied on the susceptibility to the tubercle bacilli and the probability of experimental animal.

The results are as fellows:

1. The chipmunks are more susceptible to the tubercle bacilli than the mice.

2. The chipmunks are more susceptible to the strain H^^37 Rv than the Ravenel.

3. In the chipmunks, extensive infectivity are observed in the spleen, liver and lymph nodes.

4. In the chipmunks infected with bacilli (H^^37 Rv and Ravenel), the macroscopic findings, bacterial number, Gaffky index, and histopathological findings in the spleen, liver and lymph nodes show the parallel relationship.

5. In the chipmunks infected with H^^37 Rv and Ravenel, 50 percent letal days are 22.3 days and 21 days respectively.

6. The chipmunks is most susceptible to tubercle bacilli among the known animal to date, and able to feed as an experimental animal and appropriate to an experimental animal for experiulental toberculosis.